Catalysis and function of the p38 alpha.MK2a signaling complex

The p38 mitogen-activated protein kinase (p38) pathway is required for the production of proinflammatory cytokines (TNFalpha and IL-1) that mediate the chronic inflammatory phases of several autoimmune diseases. Potent p38 inhibitors, such as the slow tight-binding inhibitor BIRB 796, have recently been reported to block the production of TNFalpha and IL-1beta. Here we analyze downstream signaling complexes and molecular mechanisms, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. Catalysis, signaling functions, and molecular interactions involving p38alpha and one of its downstream signaling partners, mitogen-activated protein kinase-activated protein kinase 2 (MK2), have been explored by steady-state kinetics, surface plasmon resonance, isothermal calorimetry, and stopped-flow fluorescence. Functional 1/1 signaling complexes (Kd = 1-100 nM) composed of activated and nonactivated forms of p38alpha and a splice variant of MK2 (MK2a) were characterized. Catalysis of MK2a phosphorylation and activation by p38alpha was observed to be efficient under conditions where substrate is saturating (kcat(app) = 0.05-0.3 s(-1)) and nonsaturating (kcat(app)/KM(app) = 1-3 x 10(6) M(-1) s(-1)). Specific interactions between the carboxy-terminal residues of MK2a (370-400) and p38alpha precipitate formation of a high-affinity complex (Kd = 20 nM); the p38alpha-dependent MK2a phosphorylation reaction was inhibited by the 30-amino acid docking domain peptide of MK2a (IC50 = 60 nM). The results indicate that the 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex, and that perturbation of the tight-docking interaction between these signaling partners prevents the phosphorylation of MK2a. The thermodynamic and steady-state kinetic characterization of the p38alpha.MK2a signaling complex has led to a clear understanding of complex formation, catalysis, and function on the molecular level.     

DEF Pocket in p38α Facilitates Substrate Selectivity and Mediates Autophosphorylation

Signaling processes are primarily promoted by molecular recognition and corresponding protein-protein interactions. One of the key eukaryotic signaling pathways is the MAP kinase cascade involved in vital cellular processes such as cell proliferation, differentiation, apoptosis, and stress response. The principle recognition site of MAP kinases, the common docking (CD) region, forms selective interactions with substrates, upstream activators, and phosphatases. A second docking site, defined as the DEF site interaction pocket (DEF pocket), is formed subsequent to ERK2 and p38α activation. Both crystal structures of p38α in its dually phosphorylated form and of intrinsically active mutants showed the DEF pocket, giving motivation for studying its role in substrate activation and selectivity. Mutating selected DEF pocket residues significantly decreased the phosphorylation levels of three p38α substrates (ATFII, Elk-1, and MBP) with no apparent effect on the phosphorylation of MK2 kinase. Conversely, mutating the CD region gave the opposite effect, suggesting p38α substrates can be classified into DEF-dependent and DEF-independent substrates. In addition, mutating DEF pocket residues decreased the autophosphorylation capability of intrinsically active p38α mutants, suggesting DEF-mediated trans-autophosphorylation in p38α. These results could contribute to understanding substrate selectivity of p38α and serve as a platform for designing p38α-selective DEF site blockers, which partially inhibit p38α binding DEF-dependent substrates, whereas maintaining its other functions intact. In this context, preliminary results using synthetic peptides reveal significant inhibition of substrate phosphorylation by activated p38α.     

Computer-aided drug design guided by hydrogen/deuterium exchange mass spectrometry: A powerful combination for the development of potent and selective inhibitors of Group VIA calcium-independent phospholipase A2

Potent and selective inhibitors for phospholipases A₂ (PLA₂) are useful for studying their intracellular functions. PLA₂ enzymes liberate arachidonic acid from phospholipids activating eicosanoid pathways that involve cyclooxygenase (COX) and lipoxygenase (LOX) leading to inflammation. Anti-inflammatory drugs target COX and LOX; thus, PLA₂ can also be targeted to diminish inflammation at an earlier stage in the process. This paper describes the employment of enzymatic assays, hydrogen/deuterium exchange mass spectrometry (DXMS) and computational chemistry to develop PLA₂ inhibitors. Beta-thioether trifluoromethylketones (TFKs) were screened against human GVIA calcium-independent, GIVA cytosolic and GV secreted PLA₂s. These compounds exhibited inhibition toward Group VIA calcium-independent PLA₂ (GVIA iPLA₂), with the most potent and selective inhibitor 3 (OTFP) obtaining an X_I(50) of 0.0002 mole fraction (IC₅₀ of 110 nM). DXMS binding experiments in the presence of OTFP revealed the peptide regions of GVIA iPLA₂ that interact with the inhibitor. Molecular docking and dynamics simulations in the presence of a membrane were guided by the DXMS data in order to identify the binding mode of OTFP. Clustering analysis showed the binding mode of OTFP that occupied 70% of the binding modes occurring during the simulation. The resulted 3D complex was used for docking studies and a structure–activity relationship (SAR) was established. This paper describes a novel multidisciplinary approach in which a 3D complex of GVIA iPLA₂ with an inhibitor is reported and validated by experimental data. The SAR showed that the sulfur atom is vital for the potency of beta-thioether analogues, while the hydrophobic chain is important for selectivity. This work constitutes the foundation for further design, synthesis and inhibition studies in order to develop new beta-thioether analogues that are potent and selective for GVIA iPLA₂ exclusively.     

The molecular basis for coxib inhibition of p38alpha MAP kinase

In this work, we present the results of two combined approaches, molecular docking and comparative molecular field analysis (CoMFA), to propose how the selective cyclooxygenase-2 inhibitor celecoxib could act as a p38 mitogen-activated protein (MAP) kinase inhibitor. The docking analysis revealed why celecoxib has a less favorable binding energy (DeltaG= -12.4kcal/mol) than the selective p38 MAP kinase (p38 MAPK) inhibitor, SB203580 (DeltaG= -22.2kcal/mol). The CoMFA results revealed unfavorable steric effects that can be related to the predicted lower p38 MAP kinase inhibitory activity of celecoxib. Additionally, FlexX and CoMFA results also suggested that etoricoxib, another selective COX-2 inhibitor, could inhibit p38 MAP kinase.     

Crystal structure of the p38 alpha-MAPKAP kinase 2 heterodimer

The p38 signaling pathway is activated in response to cell stress and induces production of proinflammatory cytokines. P38alpha is phosphorylated and activated in response to cell stress by MKK3 and MKK6 and in turn phosphorylates a number of substrates, including MAPKAP kinase 2 (MK2). We have determined the crystal structure of the unphosphorylated p38alpha-MK2 heterodimer. The C-terminal regulatory domain of MK2 binds in the docking groove of p38alpha, and the ATP-binding sites of both kinases are at the heterodimer interface. The conformation suggests an extra mechanism in addition to the regulation of the p38alpha and MK2 phosphorylation states that prevents phosphorylation of substrates in the absence of cell stress. Addition of constitutively active MKK6-DD results in rapid phosphorylation of the p38alpha-MK2 heterodimer.     

Rapid and quantitative detection of p38 kinase pathway in mouse blood monocyte

The p38 alpha mitogen-activated protein kinase (MAPK) is essential in controlling the production of many proinflammatory cytokines, and its specific inhibitor can effectively block their production for treating human diseases. To effectively identify highly specific p38 alpha inhibitors in vivo, we developed an ex vivo mouse blood cell-based assay by flow cytometry to measure the intracellular p38 alpha kinase activation. We first attempted to identify the individual blood cell population in which the p38 alpha kinase pathway is highly expressed and activated. Based on CD11b, combined with Ly-6G cell surface expression, we identified two distinct subsets of non-neutrophilic myeloid cells, CD11b(Med)Ly-6G(-) and CD11b(Lo)Ly-6G(-), and characterized them as monocytes and natural killer (NK) cells, respectively. Then, we demonstrated that only monocytes, not NK cells, expressed a high level of p38 alpha kinase, which was rapidly activated by anisomycin stimulation as evidenced by the phosphorylation of both p38 and its substrate, MAPKAP-K2 (MK2). Finally, the p38 alpha kinase pathway activation in monocytes was fully inhibited by a highly selective p38 alpha kinase inhibitor dose-dependently in vitro and in vivo. In conclusion, we demonstrated an effective method for separating blood monocytes from other cells and for detecting the expression level and activation of the p38 alpha kinase pathway in monocytes, which provided a new approach for the rapid identification of specific p38 alpha inhibitors.     

Geldanamycin Enhances Retrograde Transport of Shiga Toxin in HEp-2 Cells

The heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA) has been shown to alter endosomal sorting, diverting cargo destined for the recycling pathway into the lysosomal pathway. Here we investigated whether GA also affects the sorting of cargo into the retrograde pathway from endosomes to the Golgi apparatus. As a model cargo we used the bacterial toxin Shiga toxin, which exploits the retrograde pathway as an entry route to the cytosol. Indeed, GA treatment of HEp-2 cells strongly increased the Shiga toxin transport to the Golgi apparatus. The enhanced Golgi transport was not due to increased endocytic uptake of the toxin or perturbed recycling, suggesting that GA selectively enhances endosomal sorting into the retrograde pathway. Moreover, GA activated p38 and both inhibitors of p38 or its substrate MK2 partially counteracted the GA-induced increase in Shiga toxin transport. Thus, our data suggest that GA-induced p38 and MK2 activation participate in the increased Shiga toxin transport to the Golgi apparatus.     

Catalytically active MAP KAP kinase 2 structures in complex with staurosporine and ADP reveal differences with the autoinhibited enzyme

MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41-364, in complex with staurosporine at 2.7 A and with ADP at 3.2 A, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the K(m) for ATP is very similar for MK2 41-364 and p38-activated MK2 41-400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41-364. However, phosphorylation of MK2 41-364 by p38 restores the V(max) and K(m) for peptide substrate to values comparable to those seen in p38-activated MK2 41-400, suggesting a mechanism for regulation of enzyme activity.     

MAPK-activated protein kinase 2 deficiency in microglia inhibits pro-inflammatory mediator release and resultant neurotoxicity. Relevance to neuroinflammation in a transgenic mouse model of Alzheimer disease

MAPK-activated protein kinase 2 (MAPKAP K2 or MK2) is one of several kinases directly regulated by p38 MAPK. A role for p38 MAPK in the pathology of Alzheimer disease (AD) has previously been suggested. Here, we provide evidence to suggest that MK2 also plays a role in neuroinflammatory and neurodegenerative pathology of relevance to AD. MK2 activation and expression were increased in lipopolysaccharide (LPS) + interferon gamma-stimulated microglial cells, implicating a role for MK2 in eliciting a pro-inflammatory response. Microglia cultured ex vivo from MK2-deficient (MK2-/-) mice demonstrated significant inhibition in release of tumor necrosis factor alpha, KC (mouse chemokine with highest sequence identity to human GROs and interleukin-8), and macrophage inflammatory protein 1alpha on stimulation with LPS + interferon gamma or amyloid-beta peptide (1-42) compared with MK2+/+ wild-type microglia. Consistent with an inhibition in pro-inflammatory mediator release, cortical neurons co-cultured with LPS + interferon gamma-stimulated or amyloid-beta peptide (1-42)-stimulated MK2-/- microglia were protected from microglial-mediated neuronal cell toxicity. In a transgenic mouse model of AD in which amyloid precursor protein and presenilin-1 harboring familial AD mutations are overexpressed in specific regions of the brain, elevated activation and expression of MK2 correlated with beta-amyloid deposition, microglial activation, and up-regulation of tumor necrosis factor alpha, macrophage inflammatory protein 1alpha, and KC gene expression in the same brain regions. Our data propose a role for MK2 in AD brain pathology, for which neuroinflammation involving cytokines and chemokines and overt neuronal loss have been documented.     

MAPK-activated protein kinase-2 (MK2)-mediated formation and phosphorylation-regulated dissociation of the signal complex consisting of p38, MK2, Akt, and Hsp27

The p38 MAPK and heat shock protein 27 (hsp27) form a signaling complex with serine/threonine kinase Akt and MAPK-activated protein kinase-2 (MK2), which plays an important role in controlling stress-induced apoptosis and reorganizing actin cytoskeleton. However, regulation of the complex is poorly understood. In this study, the interaction between p38 and hsp27 was visualized in single living L929 cells using fluorescence resonance energy transfer technology, while their association with Akt was examined by immunoprecipitation analysis. Under normal growth conditions, p38 kinase constitutively interacts with hsp27. When cells were exposed to H(2)O(2) or stimulated by arachidonic acid, this interaction was disrupted. However, inhibition of the activation of p38 and Akt by selective inhibitors or overexpression of the kinase-dead mutant of p38 diminished such effects. Furthermore, mutation of phosphorylation sites of hsp27 renders the interaction resistant to H(2)O(2) and arachidonic acid. It was interesting to find that the interaction disappeared in the cells from MK2-knock-out mice or the cells treated with lemptomycin B that blocks export of MK2 from nucleus to cytosol. However, MK2 is not required for the association of hsp27 with Akt. This study suggests that MK2 mediates the incorporation of p38 into the pre-existing complex of hsp27 with Akt. Phosphorylation of hsp27 finally breaks the signaling complex.     

Structural basis for a high affinity inhibitor bound to protein kinase MK2

The Ser/Thr protein kinase MAPKAP kinase 2 (MK2) plays a crucial role in inflammation. We determined the structure of the kinase domain of MK2 in complex with a low molecular mass inhibitor in two different crystal forms, obtained from soaking and co-crystallization. To our knowledge, these are the first structures of MK2 showing the binding mode of an inhibitor with high binding affinity (IC50 8.5 nM). The two crystal forms revealed conformational flexibility in the binding site and extend the experimental basis for rational drug design. Crystal form-1 contained one MK2 molecule per asymmetric unit. Form-2 contained 12 molecules, which arrange into two different types of MK2 trimers. One of them may serve as a model for an intermediate state during substrate phosphorylation, as each MK2 monomer places its activation segment into the substrate peptide binding groove of the trimer neighbor.     

Understanding the molecular basis of MK2-p38α signaling complex assembly: insights into protein-protein interaction by molecular dynamics and free energy studies

The formation of a p38 MAPK and MAPK-activated protein kinase 2 (MK2) signaling complex is physiologically relevant to cellular responses such as the proinflammatory cytokine production. The interaction between p38α isoform and MK2 is of great importance for this signaling. In this study, molecular dynamics simulation and binding free energy calculation were performed on the MK2-p38α signaling complex to investigate the protein-protein interaction between the two proteins. Dynamic domain motion analyses were performed to analyze the conformational changes between the unbound and bound states of proteins during the interaction. The activation loop, αF-I helices, and loops among α helices in the C-lobe of MK2 are found to be highly flexible and exhibit significant changes upon p38α binding. The results also show that after the binding of p38α, the N- and C-terminal domains of MK2 display an opening and twisting motion centered on the activation loop. The molecular mechanics Poisson-Boltzmann and generalized-Born surface area (MM-PB/GBSA) methods were used to calculate binding free energies between MK2 and p38α. The analysis of the components of binding free energy calculation indicates that the van der Waals interaction and the nonpolar solvation energy provide the driving force for the binding process, while the electrostatic interaction contributes critically to the specificity, rather than to MK2-p38α binding affinity. The contribution of each residue at the interaction interface to the binding affinity of MK2 with p38α was also analyzed by free energy decomposition. Several important residues responsible for the protein-protein interaction were also identified.     

Molecular determinants that mediate selective activation of p38 MAP kinase isoforms

The p38 mitogen-activated protein kinase (MAPK) group is represented by four isoforms in mammals (p38alpha, p38beta2, p38gamma and p38delta). These p38 MAPK isoforms appear to mediate distinct functions in vivo due, in part, to differences in substrate phosphorylation by individual p38 MAPKs and also to selective activation by MAPK kinases (MAPKKs). Here we report the identification of two factors that contribute to the specificity of p38 MAPK activation. One mechanism of specificity is the selective formation of functional complexes between MAPKK and different p38 MAPKs. The formation of these complexes requires the presence of a MAPK docking site in the N-terminus of the MAPKK. The second mechanism that confers signaling specificity is the selective recognition of the activation loop (T-loop) of p38 MAPK isoforms. Together, these processes provide a mechanism that enables the selective activation of p38 MAPK in response to activated MAPKK.     

Distinct cellular functions of MK2

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK alpha and -beta, which bind to a basic docking motif in the C terminus of MK2 and which subsequently phosphorylate its regulatory sites. As a result of activation MK2 is exported from the nucleus to the cytoplasm and cotransports active p38 MAPK to this compartment. Here we show that the amount of p38 MAPK is significantly reduced in cells and tissues lacking MK2, indicating a stabilizing effect of MK2 for p38. Using a murine knockout model, we have previously shown that elimination of MK2 leads to a dramatic reduction of tumor necrosis factor (TNF) production in response to lipopolysaccharide. To further elucidate the role of MK2 in p38 MAPK stabilization and in TNF biosynthesis, we analyzed the ability of two MK2 isoforms and several MK2 mutants to restore both p38 MAPK protein levels and TNF biosynthesis in macrophages. We show that MK2 stabilizes p38 MAPK through its C terminus and that MK2 catalytic activity does not contribute to this stabilization. Importantly, we demonstrate that stabilizing p38 MAPK does not restore TNF biosynthesis. TNF biosynthesis is only restored with MK2 catalytic activity. We further show that, in MK2-deficient macrophages, formation of filopodia in response to extracellular stimuli is reduced. In addition, migration of MK2-deficient mouse embryonic fibroblasts (MEFs) and smooth muscle cells on fibronectin is dramatically reduced. Interestingly, reintroducing catalytic MK2 activity into MEFs alone is not sufficient to revert the migratory phenotype of these cells. In addition to catalytic activity, the proline-rich N-terminal region is necessary for rescuing the migratory phenotype. These data indicate that catalytic activity of MK2 is required for both cytokine production and cell migration. However, the proline-rich MK2 N terminus provides a distinct role restricted to cell migration.     

MAPKAPK2-mediated LSP1 phosphorylation and FMLP-induced neutrophil polarization

In neutrophils, the major substrate of MAPKAPK2 (MK2) is an F-actin binding protein LSP1. Studies using mutants of the two potential Serine phosphorylation sites in LSP1 C-terminal F-actin binding region indicated that the major phosphorylation site for MK2 is Ser243 in murine neutrophils (Ser252 in humans). Human phosphoLSP1 antibodies that recognize phosphoSer252 site were prepared and revealed fMLP-induced neutrophil LSP1 phosphorylation. The phosphorylation was inhibited by p38 MAPK (upstream kinase for MK2) inhibitor SB203580. The antibodies also detect LSP1 phosphorylation in murine neutrophils. Immunostaining revealed that in WT murine neutrophils phosphoLSP1 was localized in F-actin enriched lamellipodia and oriented toward the fMLP gradient while non-phosphoLSP1 failed to colocalize with F-actin. In suspension, WT neutrophils exhibited persistent F-actin polarization following fMLP stimulation, while MK2(-/-) neutrophils exhibited transient F-actin polarization. These studies suggest that MK2-regulated LSP1 phosphorylation is involved in stabilization of F-actin polarization during neutrophil chemotaxis.     

Two additive mechanisms impair the differentiation of 'substrate-selective' p38 inhibitors from classical p38 inhibitors in vitro

Background  

The success of anti-TNF biologics for the treatment of rheumatoid arthritis has highlighted the importance of understanding the intracellular pathways that regulate TNF production in the quest for an orally-available small molecule inhibitor. p38 is known to strongly regulate TNF production via MK2. The failure of several p38 inhibitors in the clinic suggests the importance of other downstream pathways in normal cell function. Recent work has described a 'substrate-selective' p38 inhibitor that is able to preferentially block the activity of p38 against one substrate (MK2) versus another (ATF2). Using a combined experimental and computational approach, we have examined this mechanism in greater detail for two p38 substrates, MK2 and ATF2.     

Double-Stranded RNA-Dependent Protein Kinase Regulates the Motility of Breast Cancer Cells

Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway.